The ability to extract biologically relevant information from the genome of Borrelia burgdorferi, the cause of Lyme disease, continues to be hampered by the lack of genetic tools to identify the biological functions of the many genes of unknown functions that it contains. We and others have adapted a number of classical microbial genetics tools to B. burgdorferi and have used them to isolate null mutants and their complemented derivatives in order to analyze the contribution of these genes to B. burgdorferi biology and virulence. No one yet has been able to produce genomic saturated libraries or to isolate conditional and conditional lethal mutants of this pathogen. The long-term goal of our research is the identification and characterization of virulence determinants of B. burgdorferi. One group of potential virulence determinants that may be involved in persistence of this organism in mammalian hosts is the adhesins and adhesin regulators mediating its adhesion to Type I collagen in the extracellular matrix. Data obtained by us during the previous project period indicated that gene expression in B. burgdorferi could be modulated in a regulated fashion in a B. burgdorferi strain constitutively expressing the Tet repressor using modified B. burgdorferi hybrid promoters containing tetO operators. This system was used with the non-antibiotic inducer anhydrotetracycline (ATc) to regulate gene expression by gene fusions and antisense RNA, and in preparing and screening antisense RNA libraries. We also found that the mariner transposon system could be used to isolate transposition mutants in B. burgdorferi chromosomal and plasmid genes. On the basis of a combination of these findings, we propose the following Specific Aims. 1) Identify and characterize novel B. burgdorferi Type 1 collagen adhesins/adhesin regulators using antisense RNA libraries, isogenic mutants for adhesins/adhesins regulators, and microarrays. 2) Characterize B. burgdorferi genes relevant to colonization of the extracellular matrix in chronically infected MyD88 knockout mice using a global genomic approach involving saturated B. burgdorferi genomic transposon libraries screened by microarrays. 3) Complete development of novel systems of ATc - regulated gene expression in B. burgdorferi to study its adhesion to the ECM. PUBLIC HEALTH RELEVANCE: Lyme disease is the most common vector-borne disease in the United States. This project will develop new molecular genetic tools for studying the function of the genes of the Lyme disease bacterium, B. burgdorferi. These tools will be used to identify genes that are potentially responsible for this organism's virulence and which could be targets for new vaccines and antibiotics against Lyme disease.